Jaundice occurs in approximately 60% of newborns. When severe, the elevated blood levels of bilirubin (hyperbilirubinemia) associated with jaundice pose a significant risk for neurological damage. Current diagnostic tests for severe neonatal hyperbilirubinemia do not correlate well with bilirubin encephalopathy in newborns, contributing to the uncertainty in the management of jaundiced infants. To address this problem, a new bilirubin assay will be developed to determine the plasma concentration of free unconjugated bilirubin (UCB), which is a better indicator of risk for neurological damage. In Phase I of this project, a UCB-specific probe able to detect physiologic and pathophysiologic levels of free UCB (UCBf) in neonatal plasma was produced from a fluorescently labeled mutant of a fatty acid binding protein (FABP). The specific aims of Phase II are to (1) confirm the accuracy of the Phase I UCB probe in neonatal plasma using blood samples from 800 neonates, (2) determine the effects of potential interference from conjugated bilirubin and bilirubin isomers produced during phototherapy, and (3) optimize the assay for clinical use through improvements in the probe properties and assay format. To accomplish the first aim, probe measurements of UCBf will be compared to clinical measurements of total serum bilirubin and peroxidase measurements of UCBf. The second aim will include the enzymatic synthesis of conjugated bilirubin for interference testing and UCBf measurements of samples treated with in vitro and in vivo phototherapy. The third aim will require new versions of the probe that have improved sensitivity, precision, and stability. To increase sensitivity, new libraries of fluorescently labeled FABP mutants that do not bind fatty acids will be generated by combinatorial mutagenesis of select residues within the FABP binding pocket, using the Phase I UCB probe as a template. Precision will be improved by adding a second fluorophore to the UCB probe to create a fluorescence ratio probe. Probe stability will be increased by altering the fluorophore-FABP conjugation chemistry. The third aim will also include the optimization of the UCBf assay for use in a high throughput format to facilitate future Phase III clinical trials to study the correlation between UCBf and bilirubin neurotoxicity. The successful outcome of this study will be a unique assay for UCBf that more accurately gauges the risk of bilirubin-mediated neurological damage in neonates and significantly improves the application of phototherapy for the treatment of hyperbilirubinemia. Current diagnostic tests for elevated plasma concentrations of bilirubin do not correlate well with bilirubin encephalopathy in newborns, which contributes to the uncertainty in the management of jaundiced infants. To address this problem, a new bilirubin assay will be developed to determine the plasma concentration of free unconjugated bilirubin, which is a better indicator of risk of neurological damage. Clinical use of this assay would enhance the care of jaundiced infants by reducing unnecessary therapy and improving the identification of infants at risk for bilirubin encephalopathy. [unreadable] [unreadable] [unreadable]